Somatic and germinal mutations can have severe impact on the fitness of multicellular organisms and offspring. The ability to study the occurrence of mutations in higher organisms has been limited by available methods rendering it difficult to address important questions such as 1) kinetics of mutation induction in relation to organ specificity, 2) mutation induction during development, 3) systemic effect of somatic mutations of age-related degenerative diseases, and 4) tumor progression from pre-neoplastic to neoplastic growth in relation to genomic stability. PhiX174 with the well characterized am3 mutation is used as a transgene to evaluate substitutions at the A:T base pair. The revertant detection follows a single burst protocol. Using this protocol mutations that are fixed in the animal can be separated from the mutations that arise from ex vivo damage in the DNA. Male mice hemizygous for the phiX insert were injected i.p. with various doses of ENU. The revertant frequency increased linearly with the ENU dose only among revertants that were fixed in the mouse (burst size bigger than 30) and not among revertants that were due to ex vivo events. Nine spontaneous and ten ENU induced mutants were sequenced. Most of the spontaneous mutants were base changes from A to G, and most of the ENU induced were A to T base changes. The phiX system is the only system where a direct distinction can be made between mutations that are fixed in animals and mutations that occur during phage DNA replication in transformed bacteria, which enhances its utility as a mammalian mutation system. - mice, transgenic, PhiX174, mutagenesis, male, germinal, neonatal